Protein synthesis
Lecture Details Rod Devenish, Week 3 MED1011, Biochemistry Lecture Content Translation requires tRNA, mRNA, activating enzymes and ribosomes. In translation, amino acids are linked together by order of mRNA codons, achieved by tRNA which carries the correct amino acids and reads the codon by complimentary base pairing. tRNAs are small, 75 to 80 nucleotides, have a 3D shape maintained by H bonds from internal base pairing. Conformation of tRNA facilitates their interaction with components of protein synthesis. Their amino acid attachment site is always at CCA at the 3' end. 3 bases called the anticodon interact with mRNA, is complementary base pairing to codon. Some codon-anticodon reactions can tolerate a mismatch at the 3' end of the codon with 5' base of the anticodon. This is called wobble, does not allow the genetic code to be ambiguous. Aminoacyl-tRNA synthases are a family of activating enzymes that carry out linking of tRNA and amino acids, forming charged tRNA, each activating enzyme is specific for one amino acid. It takes in the amino acid and energy which it uses up to 'activate' the amino acid, allowing the tRNA to bind to the enzyme and bond to the amino acid to become a charged tRNA. This charged bond provides the energy for making the peptide bond to join amino acids. Each ribosome has two subunits, large and small. L has 3 rRNAs and 45 proteins, S has one rRNA and 33 proteins. When not translating, the two subunits are separate. Protein and rRNA components are held together by ionic bonds and hydrophobic forces. Ribosomes are nonspecific for mRNAs and tRNAs. There are 3 sites for tRNA binding, codon-anticodon reactions occur at P and A site. A charged tRNA travels in the order A>P>E: A is amino acid, where the charged tRNA anticodon binds to the mRNA codon, P polypeptide where the tRNA adds its amino acid to the chain, E exit where the tRNA without its amino acid goes and leaves the ribosome. Translation begins with an initiation complex which includes the first tRNA and its amino acid, an S ribosomal subunit and an mRNA. This complex is bound to region near 5' end of mRNA. The start codon AUG designates the first amino acid in all proteins (some proteins have this trimmed and removed after synthesis). The L subunit then joins the complex. This process is directed by initiation factors which use GTP as an energy source. The L subunit catalyses breakage of tRNA and amino acid in the P site as well as peptide bond formation between this amino acid and the tRNA in the A site (peptidyl transferase activity). : Steps: a charged tRNA enters the A site, releases methionine and dissociates from the ribosome, opening the P site and binding the L subunit. The second tRNA then moves to the P site opening the A site. The next tRNA moves to the A site. The peptide chain is then transferred to the tRNA occupying the P site. Elongation factors assist this process. The ribosome moves down mRNA in a 5' to 3' direction. Polypeptides grow from the N terminus to the C terminus. When a stop codon is reached a release factor enters the A site instead of an amino acid. This causes translation to terminate and the completed protein to be released. The ribosome then breaks apart. Some antibiotics target transcription, as there are different prokaryotic and eukaryotic ribosomes. In a poly(ribo)some, more than one ribosome moves down the mRNA at a time. Allows simultaneous translation. Signals in the amino acid sequences direct them to their intended cellular destination. Protein synthesis occurs on free ribosomes in the cytoplasm. Information in the amino acid sequence determines one of two outcomes: finish and release to organelles; or stall translation and go to ER and finish synthesis there (proteins destined for Golgi, ER, lysosomes, outside the cell). They enter ER by interaction of a hydrophobic cell signal with a channel in the membrane, the signal sequence is recognised and then cleaved (translation occurs on the membrane itself). Some signals cause proteins to be parts of membranes, proteins with no signal go through ER and GA and into vesicles to leave the cell. Some created proteins fold spontaneously, some need chaperones. Posttranslational events include proteolysis, glycosylation (addition of sugars), phosphorylation (addition of phosphate groups). Readings Life; 304-313=